Figure 1.

ZNF598 Engages a Sub-population of Poly(A)-Stalled Ribosomes In Vitro

(A) Strategy to analyze poly(A)-stalled ribosome-nascent chain complexes produced by in vitro translation (IVT). VHP is a small autonomously folding three-helix bundle from the villin head piece.

(B) Poly(A)-stalled translation complexes were affinity purified via the nascent chain and separated by sucrose gradient sedimentation. The affinity-purified products (input) and each gradient fraction were analyzed by autoradiography (to detect nascent chains) or immunoblotting for recombinant ZNF598 or ribosomal proteins uL2 and eS24. Mono- and di-ribosome fractions are indicated.

(C) Poly(A)-stalled translation complexes from reactions lacking or containing 5 nM FLAG-tagged ZNF598 were immunopurified via the FLAG tag, and the nascent chains were detected by autoradiography. 1° and 2° indicate the position of nascent chains from the first and second ribosome of the stalled complexes (see diagram).

(D) The input and affinity-purified samples prepared as in (C) were separated by sucrose gradient and the nascent chains detected by autoradiography. The graph below the autoradiograms depicts the distribution of nascent chains corresponding to mono-ribosomes (1°, black) or di-ribosomes (2°, red) in the input sample or the nascent chains recovered by affinity purification (”IP ZNF598” - blue).

See also Figure S1.